Nasopharyngeal Staphylococcus aureus colonization among HIV-infected children in Addis Ababa, Ethiopia: antimicrobial susceptibility pattern and association with Streptococcus pneumoniae colonization

Background. Staphylococcus aureus and Streptococcus pneumoniae are common inhabitants of the nasopharynx of children. HIV-infected children have higher risk of invasive diseases caused by these pathogens. With widespread use of pneumococcal conjugate vaccines and the emergence of methicillin-resistant S. aureus , the interaction between S. aureus and S. pneumoniae is of a particular significance. We sought to determine the magnitude of colonization by methicillin-sensitive and -resistant S. aureus and colonization by S. pneumoniae ; associated risk factors and antimicrobial susceptibility pattern among HIV-infected children in Addis Ababa, Ethiopia. Method. A prospective observational study was conducted among 183 HIV-infected children at ALERT hospital Addis Ababa, Ethiopia from September 2016 to August 2018. S. aureus and S. pneumoniae were identified using standard bacteriological techniques, antimicrobial susceptibility testing was performed on S. aureus and screening for methicillin resistance was carried out by amplifying the mecA gene. Risk factors were analysed by using binary logistic regression. Results. The prevalence of nasopharyngeal S. aureus , MRSA and S. pneumoniae colonization were 27.3, 2.7 and 43.2 %, respectively. Multivariable analysis indicated an inverse association between S. aureus and S. pneumoniae nasopharyngeal colonization [aOR, 0.49; CI, (0.24, 0.99); P=0.046]. The highest level of resistance in both methicillin-sensitive S. aureus (MSSA) and MRSA was observed against tetracycline. Conclusions . We found an inverse association between S. aureus and S. pneumoniae colonization among HIV-infected children. Continued assessment of the impact of pneumococcal conjugate vaccines and antiretroviral therapy on nasopharyngeal bacterial ecology is warranted.

anterior nares, skin, perineum and pharynx.However, the most frequent carriage site for S. aureus, with a 27 % carriage rate among adults, is the nose and a causal relationship exists between S. aureus colonization and infection [2].Compared to adults, persistent carriage is mostly seen in children and carriage rates vary between 45 % in the first 8 weeks and decline to 21 % by 6 months of age [3].
Methicillin-resistant Staphylococcus aureus (MRSA) was discovered in 1961, soon after the introduction of methicillin in clinical isolates and then in the 1980s in the community.It has since spread globally and its prevalence is increasing resulting in increased health-care costs, morbidity and mortality [1,4].HIV-infected patients, due to their weakened immune system, are at increased risk for several infections, including those caused by S. aureus and MRSA [5].The morbidity and mortality associated with MRSA in patients with HIV infection [6] makes the study of MRSA colonization among the HIV-infected patients of particular interest.In addition, colonization with MRSA has been associated with a fourfold increase in the risk of infection [7].
Studies indicate that compared to healthy individuals, the prevalence of MRSA nasal colonization is higher in HIV-positive people [8,9].The risk of MRSA colonization often mirrors that of infection and additional people with higher risk of colonization/infection are, children, those in prisons, military recruits, those in poor neighbourhoods, livestock workers, individuals with prior MRSA infection and hospitalization and those with cystic fibrosis [1].The main risk factors for colonization and infection among HIV-infected patients on the other hand are use of antimicrobials, previous hospitalization and low CD4 +T lymphocyte counts [10].
MRSA is now considered an urgent threat to public health and among the priority list of pathogens for which new antibiotics are required [11].In order to make effective treatment of patients with disease due to MRSA and to prevent further transmission, accurate detection of methicillin resistance is of the utmost importance [12].
The mecA gene is highly conserved in staphylococcal strains and thus is a useful marker and is considered as the gold standard for identifying methicillin resistance in S. aureus isolates.The mecA gene is located on the staphylococcal chromosome cassette mec and encodes penicillin binding protein 2 a (PBP2a) [12].Other chromosomal factors such as femA and femB are also associated with the expression of methicillin resistance [13].
Pneumococcal conjugate vaccines (PCVs) began being introduced in infant immunization schedules from the year 2000 and studies indicate that administration of PCVs to infants modifies not only the carriage of Streptococcus pneumoniae but also that of S. aureus and there is an inverse relationship between the carriage of the two bacteria [14,15].There was also an earlier clinical trial that indicated an increase in acute otitis media due to S. aureus after PCV vaccination [16].We have recently been able to show that 5-6 years after introduction of PCV10 in Ethiopia, S. aureus was the main cause of bacteremic community-acquired pneumonia among children [17].
Earlier studies in HIV-infected children indicated a lack of association between S. pneumoniae and S. aureus carriage and cite suboptimal adaptive immunity as a possible reason [14] but there are suggestions that S. pneumoniae -S.aureus interference is CD4 + T cell-mediated and returns to normal following antiretroviral therapy (ART) [18].
In Ethiopia, PCV10 was introduced in October 2011 as a three-dose primary series (3P+0) without any booster dose and a decision has now been made to switch to PCV13.A previous study among HIV-infected children in Northern Ethiopia indicated a pharngeal carriage prevalence of 29 % for S. aureus and 12.3 % for S. pneumoniae [19].There is however a scarcity of data in Ethiopia on the relationship between the nasopharyngeal carriage of S. pneumoniae and S. aureus and the possible impact of PCV introduction especially among HIV-infected children.The aim of this study was therefore to determine the magnitude of methicillin-sensitive and -resistant S. aureus nasopharyngeal colonization and their association with S. pneumoniae colonization among HIV-infected children in Addis Ababa, Ethiopia.

Study design and setting
A prospective observational study was conducted at the All Africa Leprosy Rehabilitation and Training Hospital (ALERT), a governmental referral hospital, located in Addis Ababa, Ethiopia.The target population of this study was HIV-positive children aged 0-15 years coming for ART follow-up to the ALERT paediatric HIV clinic from 1 September 2016 to 31 August 2018.

Patient enrollment and data collection
Trained research nurses approached the parents or guardians of the HIV-infected children who came for an initiation or followup visit at the ART clinic and briefed them on the information that has been provided in the patient information sheet.Children of parents who gave consent were then included in the study.Study participants' demographic and clinical data including information on risk factors for colonization and disease were obtained and transferred to the questionnaire prepared for the study.
The dependent variable assessed was colonization with S. aureus.The independent variables assessed were gender, age, number of household members, number of rooms in the house, presence of siblings <5 years of age, parents' level of education, monthly income, exposure to cigarette smoke, reasons for hospital visit, antiretroviral treatment status, PCV vaccination,

Culture and identification
The samples were initially thawed and 100 -µl of the NP swab sample was plated on a blood agar plate (BAP) with and without gentamicin by streaking the sample using a sterile loop.BAP plates were then incubated at 37 °C with ~5 % CO 2 for 18-24 h. S. aureus was identified using colony morphology on sheep blood agar, gram staining, catalase activity, production of coagulase and growth on mannitol salt agar.Identification of S. pneumoniae was made on the basis of colony morphology, optochin susceptibility and bile solubility.

Detection of mecA genes
DNA extraction was performed by boiling S. aureus pellets in 300 µl of TE buffer as described previously [20].Detection of 310 bp fragment of mecA was performed using primer pairs: 5′-GTAG AAAT GACT GAAC GTCC GATAA-3′ and 5′ CCAA TTCC ACAT TGTT TCGG TCTAA −3′ as described previously [21].The reaction mixture contained 12.5 µl of hot star master mix (Qiagen, Hilden, Germany), 0.5 µl each of the forward and reverse primers, 9 µl of molecular grade water and 2.5 µl of the template with a final volume of 25 µl.Amplification was carried out with 40 cycles of initial heat activation at 95 °C for 15 min, denaturation at 94 °C for 30 s, followed by annealing at 52 °C for 45 s, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min.The PCR products were analysed by electrophoresis on a 2 % agarose gel.

Statistical analysis
Data were initially entered in ReDCap (Vanderbilt University, Nashville, TN, USA), exported into Excel and analysed using PASW Statistics 20 software (SPSS, Chicago, IL, USA).Sociodemographic, environmental and clinical characteristics were analysed using descriptive statistics.Bivariate analysis, using binary logistic regression, was initially performed in order to determine factors associated with S. aureus colonization.Multivariate logistic regression was then used to assess independent associations for variables significant at P< 0.1.Variables at P<0.05 were then considered statistically significant in the multivariable analysis.

Ethical considerations
The study procedures were in accordance with the Helsinki Declaration.The study protocol was approved by the AHRI/ALERT Ethical Review Committee (AAERC) (PO/017/2015).An 0fficial permission letter was obtained from the ALERT hospital.A written informed assent and consent was obtained from study participants, and parents or guardians of children, respectively, before including them in the study.The study participants' right to refuse or not give nasopharyngeal samples without affecting their routine medical services was granted.Samples were coded to keep the confidentiality of the study participants' personal information.

Socio-demographic and clinical characteristics of the study participants
Out of the potentially eligible 750 HIV-infected children, nasopharyngeal samples were collected from a total of 183 HIVinfected children at the ALERT hospital.Among the study participants, 50.8 %(93/183) were girls; the mean age of the study  1).

Prevalence of S. aureus and S. pneumoniae colonization
The overall prevalence of S. aureus nasopharyngeal colonization among the HIV-infected children in this study was 27.3 %(50/183) whereas.S. pneumoniae was isolated from 43.2 %(79/183) of the children (Table 1).

Prevalence of mRSA colonization and detection of mecA
mecA was detected in 10 %(5/50) of the S. aureus isolates and MRSA colonization rate was therefore 2.7 %(5/183).

DISCuSSIon
Nasopharyngeal colonization and infection with S. aureus and MRSA is higher in HIV-infected children than those without HIV [22,23].Determining the S. aureus carriage rate and antibiotic resistance profiles is important in order to identify risk factors associated with S. aureus infection.
In the current study, we found that 5-7 years after introduction of PCV10 in Ethiopia, prevalence of S. aureus nasopharyngeal carriage was 27.3 %(50/183).Our result is in agreement with a similar study done in Northern Ethiopia 29 %(88/300) [19].Similar prevalence has also been reported from South Africa, prior to the introduction of PCVs 25.6 %(91/355) [24].Our finding was however lower than a report from results from a study in Brazil (45.16 %) before the introduction of PCVs [23].This might be because of geographical differences, sample size, the use of antiretroviral drugs and the introduction of PCVs.Our results were however higher than S. aureus nasopharyngeal carriage prevalence among HIV-uninfected under-5 children reported from Uganda, 19.4 %(144/742) [25] and Ghana, 23.2 %(95/210) [26].This might be due to the difference in the age group as most (70 %) of the children in the current study are aged 10-14 years.
Our finding indicates that the prevalence of MRSA nasopharyngeal colonization in HIV-infected children was 2.7 %(5/183).
Our finding is similar to nasal MRSA colonization reported among children and adults in Northern Ethiopia 2.4 %(6/249) [27].Our findings were however lower than the pharyngeal colonization reported in a similar group of HIV-infected children in Northern Ethiopia 9.7 %(29/300) [19].In the study by Mulu et al. and colleagues, methicillin resistance was determined using cefoxitin discs whereas in the current study, we used mecA amplification to determine methicillin resistance.Although Cefoxitin resistance is known as a reliable surrogate marker for mecA-mediated methicillin resistance [28], there are studies that have questioned its accuracy [29].Additional reasons for discrepancies in prevalence of MRSA could be differences in study locations and periods, differences in the implementation of infection prevention among the hospitals and rational antibiotic use practices in the two settings.In addition, in both studies, it is important to note that the reported prevalences are of the study cohorts and do not necessarily represent the overall situation in the country.
In this study, nasopharyngeal carriage of S. pneumoniae was 43.2 %(79/183).The results are higher than pharyngeal carriage reported among similar group of children in Northern Ethiopia 31(10.3%) [27].The study by Mulu and colleagues from Northern Ethiopia reports pharyngeal carriage and samples might not have been taken from the nasopharynx.Our results were however lower than those reported from the same hospital as the current study at a later time point (2018-2019), 52 %(26/50) [30].The differences in carriage prevalence might be due to differences in the median ages of the study populations, which was the 53.5 months' range  in the study by Lemma and colleagues while in the current study the median age is the 132 months' range .Our results were also similar to those reported among HIV-uninfected children of a similar age range in Southern Ethiopia [31].
In multivariable analyses, there was an inverse association between S. aureus and S. pneumoniae nasopharyngeal colonization in this group of HIV-infected children 5 to 7 years after introduction of PCV10 in the country.Accordingly HIV-infected children who are colonized with S. pneumoniae are less likely to be colonized with S. aureus.A negative association between carriage of S. aureus and PCV7 vaccine type S. pneumoniae was first reported close to two decades ago by two studies from Israel [32] and the Netherlands [33] before the introduction of pneumococcal conjugate vaccines.Since then, other studies from different parts of the world have reported similar results [34,35] mainly among healthy or HIV-uninfected children.
However, studies among HIV-infected children from South Africa indicated lack of association between the nasopharyngeal colonization of S. aureus and S. pneumoniae [14,24].The possible reasons for the lack of competition between the two bacteria in HIV-uninfected children included reduced mucosal immunity and therefore decreased immunological pressure and increased exposure to respiratory pathogens [24].Other authors however suggested that a secondary hostrelated mechanism associated mainly with CD4 + T cells might play an important role in the pathway of interaction between S. pneumoniae and S. aureus [36].The studies from South Africa did not study the correlation between CD4 + T cell counts and bacterial colonization or interaction.In the current study, the median (IQR) CD4 +T cell count of the study participants was 732 (524-924) cells/mm 3 .
According to the Ethiopian national guideline for HIV prevention, care and treatment, for all children living with HIV, ART is given as early as possible regardless of their WHO clinical stages and CD4 + T cell counts/percentage [37].Studies indicate that early HIV diagnosis and early ART reduce infant mortality and HIV progression [38].In addition, in HIV-infected infants with early ART treatment CD4 + T cell counts stabilize at the highest levels possible [39].We therefore hypothesize the inverse interaction between S. aureus and S. pneumoniae seen in the HIV-infected children in this study, similar to previous reports on the inverse interaction between S. aureus and S. pneumoniae in HIV-uninfected children, is due to the impact of ART treatment.
People living with HIV are at increased risk of acquisition and infection with drug-resistant bacteria [6].In this study, among both MSSA and MRSA isolates, the highest level of resistance was seen against tetracycline, which was consistent with a similar study among HIV-infected children in Northern Ethiopia [40] although a higher percentage of resistance to tetracycline among MRSA isolates (72 %) compared to our study (35.2 %) were reported.The low rates of resistance to most of the antibiotics tested observed in this study might suggest that most of the MSSA and MRSA were community-acquired strains [41].
Our study had some limitations.Firstly, prevalence of S. aureus colonization might have been underestimated since we sampled only the nasopharynx.Since our results originate from only one hospital, the results might not be fully representative of children in the whole of Addis Ababa or Ethiopia.Because serotyping was not performed for the S. pneumoniae isolates, we could not determine the relationship between vaccine type and non-vaccine type S. pneumoniae colonization and S. aureus colonization.In addition, our focus was only on colonization and the study was not designed to identify disease caused by these pathogens.

ConCLuSIonS
The results of this study suggest that 5 to 7 years after introduction of PCV10 in Ethiopia and more than 12 years after introduction of ART in Ethiopia, the nasopharyngeal colonization rate of S. aureus in HIV-infected children at ALERT hospital, Addis Ababa, Ethiopia was 27.3 and 10 % of the S. aureus isolates were MRSA.In addition, there was an inverse relationship between the colonization of S. aureus and S. pneumoniae.

Please rate the quality of the presentation and structure of the manuscript Satisfactory
To what extent are the conclusions supported by the data?Strongly support

If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines? Yes
SciScore report https://doi.org/10.1099/acmi.0.000557.v2.1 • As per your recommendation, level has now been replaced with rate.

Please provide a definition for MDR in this context
• A definition for MDR has now been given on line 162-163.

How the style and organisation of the paper communicates and represents key findings
Clear tables present the observations clearly and there is a good flow of findings and discussion

Literature analysis or discussion
A key prior publication of this problem in this setting is not mentioned in the discussion (where a lack of prior information is cited as a motivating factor for the study -line 109), but is cited in discussion as a comparison (line 264).While the existence of prior work does not negate the value of this, it's hard to reconcile those two statements.I would encourage the authors instead to acknowledge this prior literature in the introduction and use the discussion to make clear the additional or contrary results provided by this study (as they do regarding MRSA) • Our intention of using the word scarcity was to indicate that there were only few studies.However as per your suggestion, we have now cited and acknowledge previous study in the introduction.
The discussion on different observations between S. aureus and S. penumoniae in children living with HIV with and without ART is informative and clear

Any other relevant comments
The authors could consider including in discussion what they see as the clinical implications of these findings.
• With low rate of MRSA carriage it will be misleading to describe clinical implications of S. aureus carriage.However, the similarity between these groups of children on ART with HIV-uninfected children indicates the positive impact of ART and this has been already been discussed.• A correction has now been made as per your suggestion.
-line 72 -cited reference 5 does not appear to establish the claim that HIV increases risk of S. aureus infection • Thank you for pointing this out.This has now been replaced with the correct citation.aureus and Streptococcus pneumoniae and the associated risk factors and S. aureus antimicrobial susceptibility pattern 5-6 years after the introduction of the pneumococcal conjugate vaccine (PCV10) in Ethiopia were also investigated.There is a lack of data on the colonisation rates in this population and therefore this study provides valuable information to the field.There are however, some points for the authors to consider/address in order to improve the presentation of their findings in this manuscript.
• Thank you.The suggested amendment has been made 2. The data presented in Table 1 is difficult to interpret.
a. What is the denominator in the "Colonized with S. aureus" column?
b. Please explain what the percentages (%) in the "Colonized with S. aureus" column refer to.
• Your comments are well, taken.Based on similar suggestions from reviewer 1 too, a correction has now been made.The initial percentages were totally incorrect and it probably originated during transfer from SPSS to the manuscript whereby values from a totally different table were transferred.The revised percentages now indicate the percentage share within S. aureus colonization.
3. Table 1 appears to correctly list the percentage of pneumococcal colonisation as 43.2% (79/183) however, line 234 lists the percentage as 43.3%.Please explain the reason for this?
• It was just a typing error and a correction has now been made.
4. Table 2.The legend does not include the definition for cOR.
• It is now included 5. Lines 247-253 -What are the values (numerators and denominators) used to calculate these percentages?
• The numerators and denominators have now been included.
6. Line 255 -Repeated use of the word "of ".
• Incorrectly used words have now been removed 7. Line 263 -please clarify that this reported prevalence was observed in the study cohort and not necessarily the entire population of Ethiopia.
• A statement that describes this has now been included 8. Lines 267-268 -Suggestion to expand on the differences noted.For example, the increased vs decreased used of antiretroviral drugs, what types of PCV vaccine were used and the vaccine uptake rates.
• Available additional information has now been included 9. Line 291-294 -Are you implying that "HIV-uninfected" children have reduced mucosal immunity compared to those infected with HIV?I am not sure that this is what the referenced publication is stating.Please can you clarify this statement.
• The statement is about the interaction between S. aureusand S. pneumoniaecarriage and not about mucosal immunity.A clarification has now been provided.
10. Please include the range of the CD4+ T cell counts observed in the study participants.
• The median and IQR of the CD4+ T cell count has now been included.
11. Line 307 -Although not presented in this manuscript, is there a correlation between the CD4+ T cell counts and the carriage rates in this study cohort to support this theory?If so it would be useful to include these findings.
• We found no correlation between either the mean value or the categorized value of the CD$+ T cell count and the carriage of either S. aureus or S. pneumoniae.As we can see from the median (IQR) value, because most of the children have been on ART for a long time the median CD4 count is high.
12. Line 318 -"since we sampled only sampled nasopharyngeal swabs" please clarify this sentence.
• It has been corrected as 'we sampled only the nasopharynx' .It is meant to indicate that we did not also sample the nose is the case in many studies for S. aureus colonization.
and the vaccine uptake rates.9. Line 291-294 -Are you implying that "HIV-uninfected" children have reduced mucosal immunity compared to those infected with HIV?I am not sure that this is what the referenced publication is stating.Please can you clarify this statement.10.
Please include the range of the CD4+ T cell counts observed in the study participants.11.Line 307 -Although not presented in this manuscript, is there a correlation between the CD4+ T cell counts and the carriage rates in this study cohort to support this theory?If so it would be useful to include these findings.12.
Line 318 -"since we sampled only sampled nasopharyngeal swabs" please clarify this sentence.13.
Lines 319-320 -"the results might be fully representative of children in the whole of Addis Ababa or Ethiopia" why does this study cohort fully represent the children in the whole of Addis Ababa or Ethiopia?14.
Line 328 -Do you mean the nasopharyngeal colonisation rates of S. aureus or total colonisation based on all the bacteria isolated in this study.15.Please include a description on how the MSSA, MRSA and pneumococcal carriage rates observed in the HIV infected children in this study compare to those reported in studies of HIV-uninfected children in Ethiopia and similar populations.

Please rate the quality of the presentation and structure of the manuscript Poor
To what extent are the conclusions supported by the data?Partially support Comments: 1. Methodological rigour, reproducibility and availability of underlying data This is an observational cohort of HIV infected children, reviewing frequency of colonisation of MSSA, MRSA and S. pneumoniae.The authors aim to report prevalence in HIV infected children in the post PCV period, and the study design chosen is appropriate to this aim.Study is conducted with well described methods.In Methods, sample size paragraph implies this was designed to provide a comparison to another observational study of S. aureus nasal carriage in Uganda post PCV rollout.If designed as a comparison so, this should be made explicit and results presented.However if no comparison is being made, this sample size calculation can be omitted.Associated clinical details collected, giving a thorough list of possible RF.Authors should include a description as to how missing data is handled?Microbiological methods are appropriate and ethical concerns given due concern and adequately addressed.2. Presentation of results Table 1 -Reason for hospital visit appears to have missing data; please ensure all missing data is clear (eg state for how many the observation is not available Table 1 -% figures given for various observations in those with S. aureus appear to be incorrect.eg S.pneumo carraige 15/50 Yes, but % given is 21%, and 35/50 given as no, % given is 28.4 (should be 70%) LRTI in last 3 months 2 of 50 answer yes, % given is 1.6%; 48/50 answer given is now, % given is 48.4 Please review all % calculations in this table -this is the major revision required, and without being certain that the numbers provided are accurate, can't state that the conclusions are supported by data Please also add the total number to the top row for "Colonised with S. aureus" to make this clear In presenting data on antimicrobial susceptibility, care needed with the term 'level', which is often used to refer to the MIC compared with cut off (eg 'high level gentamicin resistance' in Enterococci).For avoidance of confusion, suggest use of 'rate' when describing the proportion of isolates with observed resistance.Please provide a definition for MDR in this context 3. How the style and organisation of the paper communicates and represents key findings Clear tables present the observations clearly and there is a good flow of findings and discussion 4. Literature analysis or discussion A key prior publication of this problem in this setting is not mentioned in the discussion (where a lack of prior information is cited as a motivating factor for the study -line 109), but is cited in discussion as a comparison (line 264).While the existence of prior work does not negate the value of this, it's hard to reconcile those two statements.I would encourage the authors instead to acknowledge this prior literature in the introduction and use the discussion to make clear the additional or contrary results provided by this study (as they do regarding MRSA) The discussion on different observations between S. aureus and S. penumoniae in children living with HIV with and without ART is informative and clear 5.

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes

2 3.
Shet A, Mathema B, Mediavilla JR, Kishii K, Mehandru S, Jeane-Pierre P, et al.Colonization and subsequent skin and soft tissue infection due to methicillin-resistant Staphylococcus aureus in a cohort of otherwise healthy adults infected with HIV type 1. J Infect Dis.2009;200: 88-93.doi:10.1086/599315Reviewer Please rate the manuscript for methodological rigour Reviewer 2: Satisfactory Please rate the quality of the presentation and structure of the manuscript Reviewer 2: Poor To what extent are the conclusions supported by the data?Reviewer 2: Partially support Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?Reviewer 2: No: If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Reviewer 2: Yes: Reviewer 2 Comments to Author: This manuscript describes the investigation of the nasopharyngeal carriage rates of Staphylococcus aureus in HIV infected children attending All Africa Leprosy Rehabilitation and Training Hospital (ALERT) paediatric HIV clinic in Addis Ababa, Ethiopia.The magnitude of colonization by methicillin-sensitive and -resistant S.

Reviewer 1
recommendation and comments https://doi.org/10.1099/acmi.0.000557.v1.3 © 2023 Young B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Bernadette Young; University of Oxford Nuffield Department of Clinical Medicine: University of Oxford Nuffield Department of Medicine, UNITED KINGDOM https://orcid.org/0000-0001-6071-6770Date report received: 08 March 2023 Recommendation: Major Revision Any other relevant comments The authors could consider including in discussion what they see as the clinical implications of these findings.Minor comments -lines 67-68 rephrasing to the 1960's (or replace with 1961); and I understand CA-MRSA reported as early as the 1980s David MZ, Daum RS.Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic.Clin Microbiol Rev. 2010 Jul;23(3):616-87.doi: 10.1128/CMR.00081-09.PMID: 20610826; PMCID: PMC2901661.-line 72 -cited reference 5 does not appear to establish the claim that HIV increases risk of S. aureus infection Please rate the manuscript for methodological rigour Good Please rate the quality of the presentation and structure of the manuscript Good To what extent are the conclusions supported by the data?Partially support

Table 1 .
Socio-demographic, environmental and clinical characteristics and their association with S. aureus colonization among HIV-infected children aged <15 years at ALERT hospital, Addis Ababa, Ethiopia Continued participants was 10.78±2.68 years.The median [interquartile range (IQR)] CD4 +T cell count of the study participants was 73(524-924) cells/mm 3 and 95.6 %(175/183) of the children have received full dose of PCV10 (Table

Table 2 .
Risk factors associated with S. aureus colonization among HIV-infected children aged <15 years at ALERT hospital, Addis Ababa, Ethiopia Thank you for addressing reviewer comments.However there are still some calculation errors in the manuscriptplease check all % calculations again before the manuscript goes through final processing.
Reviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000557.v2.3 © 2023 Young B. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Bernadette Young; University of Oxford Nuffield Department of Clinical Medicine: University of Oxford Nuffield Department of Medicine, UNITED KINGDOM https://orcid.org/0000-0001-6071-6770Date report received: 23 July 2023 Recommendation: Accept Comments: The authors have addressed concerns in first review, and the results are now well situated within the relevant literature.Minor comments Table 1 male 90/183 is given at 72.2%.Please re-check all percentage calculations very carefully.spelling pharyngeal on line 109 remove apostrophe from prevalences line 271